Method for limiting damage due to bacteriophages in fermentation media

ABSTRACT

Method for limiting damage resulting from the presence of bacteriophages in fermentation medium by effecting fermentation in the presence of at least one N-acylamino derivative of glutamic acid, glutamine or homocysteic acid or metallic salts thereof wherein the acyl group is characterized by containing an alkyl or alkenyl moiety having from about 16 to 18 carbon atoms.

United States Patent Yamanaka et al.

1451 Apr. 29, 1975 METHOD FOR LIMITING DAMAGE DUE TO BACTERIOPHAGES INFERMENTATION MEDIA Inventors: Shigeru Yamanaka, Yokohama;

Nobukazu Kashima, Kawasaki; Koji Mitsugi, Yokohama, all of JapanAssignee: Ajinomoto Co., Inc., Tokyo, Japan Filed: Sept. 3, 1974 Appl.No.: 503,047

Foreign Application Priority Data Sept. 4, 1973 Japan 48-99635 US. Cl195/122; 195/47 Int. Cl. ..C12b l/24; C12d 13/06 Field of Search195/100, 101, 102, 110,

References Cited FOREIGN PATENTS OR APPLICATIONS 45-32234 10/1970 Japan195/122 Primary Examiner-A. Louis Monacell Assistan! Examiner-C. A. Fan

Attorney, Agent, or Firm-Cooper, Dunham, Clark, Griffin & Moran [57]ABSTRACT 8 Claims, No Drawings METHOD FOR LIMITING DAMAGE DUE TOBACTERIOPI-IAGES IN FERMENTATION MEDIA BACKGROUND OF THE INVENTION Thepresence of bacteriophages in fermentation media designed to produceimportant products such as drugs, vitamins, amino acids and the like isa serious problem which has plagued the industrial production of theseimportant materials by limiting the growth of desirable bacteria ingrowth cultures. Accordingly, much effort has been directed to theproblem of controlling these bacterial viruses.

THE INVENTION A method has now been discovered for limiting the damageresulting from the presence and growth of bacteriophages in fermentationmedia by effecting the fermentation in a medium containing an amount ofat least one selected N-acylamino derivative of glutamic acid, glutamineor homocysteic acid which is effective to inhibit the production of thebacteriophage. The N- acylamino group is characterized as one whichcontains an alkyl or alkenyl moiety having from about 16 to 18 carbonatoms. Metallic salts of these compounds may also be employed, thepreferred being alkali metal salts, especially sodium or potassiumsalts.

A particular advantage of the invention is that. while the selectedagents inhibit propagation of the bacteriophages they have little or noeffect on the growth of the bacteria or the production of the desiredproduct of the fermentation.

The inhibiting agent can be added to the medium prior to the initiationof bacterial growth, or after such growth is already well underway.Normally, it is added in the initial stages of fermentation or at leastbefore the growth of bacteria reaches the stationary phase. The amountof the selected compound or compounds employed is typically less than 50u/ml, and there is rarely any advantage in exceeding this amount. It isgenerally preferred to use more than 2 palm] of N- acylamino acid. It isnot necessary to alter the usual methods of bacteia cultivation due tothe presence of the inhibiting agent.

The advantage of this invention are applicable to fermentation processesincluding, for example, those designed for the production ofantibiotics, enzymes, amino acids, nucleotides and nucleosides.

The following non-limiting examples are given by way of illustrationonly.

EXAMPLE 1 An aqueous culture medium was prepared to contain, perdeciliter, 3.6 g glucose, 0.1 g KI-I PO 0.1 g MgSO -7H O, 2 mg FeSO'7l-I O, 2 mg MnSO '4I-I O, 24 mg (as nitrogen) soyprotien acidhydrolyzate, 100 pg thiamine-HCl, 0.3 pg biotin. N-acylamino acid shownin Table l (50 #g/ml) was also added to the medium.

Brevibacterium lactofermentum ATCC 13869 was inoculated into 30 ml ofthe aqueous medium placed in 500 ml flask, to a concentration of 2.74 Xcells/ml, and the medium was infected with phage L, (Journal of theAgricultural Chemical Society of Japan, Vol. 37, No. 1], 686-689 (1963)at a concentration of 1.16 X 10 P.F.U./ml.

LII

I Cultivation was carried out at 30C for 24 hours with shaking. Glutamicacid accumulated in the cultivation broth is shown in Table 1.

Table 1 Glutamic acid accumulated g/dl N-acyl amino acid added N-acylmoiety. amino acid moiety Lauric acid Myristic acid Palmitic acidStcaric acid Olcic acid Arachidic acid Lauric acid Myristic acidPalmitic acid Stearic acid Oleic acid Arachidic acid Laurie acidHomocysteic ac id Glutamic acid Myristic acid Palmitic acid Stearic acidOleic acid As a comparison test, when the fermentation was carried outwithout adding either N-acyl amino acid or phages, 1.63 g/dl glutamicacid accumulated, while when the fermentation was carried out in thepresence of infecting phages but without adding 50 ug/ml N-acyl aminoacid, only 0.23 g/dl glutamic acid accumulated.

EXAMPLE 2 In the process of glutamic acid fermentation as described inExample 1, N-acylamino acid as shown in Table 2 was used (50 ug/ml), andcultivation was carried out by the same manner as in Example 1. Glutamicacid accumulated in the fermentation broth is shown in Table 2.

Table 2 Glutamic acid accumulated g/dl N-acyl amino acid added N-acylmoiety. amino acid moiety N-acylamino acid as shown in Table 3 was addedto the medium of Example 1 in the amount shown in Table 3. Cultivationof Brevibacterium lactofermentum ATCC 13869 was carried out by the samemanner as in Example 1. The amounts of glutamic acid shown in Table 3were accumulated.

Table 3 Table 5-Continued Amount N-oleoyl Nstcaroyl N-palmitoyl PhageBiotin N-palmitoyl Growth Glutamic acid added glutamic acid glutaminehomocystcic glutamic acid accumulated acid 5 (#g/l) (Mg/ml) (g/ (r g/ml)1.551001) .21 (g/dl) 0.21 (g/dl) 0 a .89 0.99

3 50 0.940 1.16 g8 12 12-? {2 Infected 5 50 0.920 1.65 70 1.21 1.18 1.05l 50 0980 100 1.01 0.98 0.75 0 200 0.70 0.50 0.48

Table 6 EXAMPLE 4 Phage Biotin N-palmitoyl Growth Glutamic acid glutamicacid accumulated Brevibacterium lactofermentum ATCC 13869 was (#g/l)(pg/ml) 1/26) (g/dl) inoculated into the medium of Example 1 (2 X 10 O 04O cells/ml) which was infected at an initial concentration I 0 8: 8{:23 of 2 X 10 phages/ml. N-acylamino acid mixtures con- Not 2 0 0.9501.23 taining 50 percent N-oleoyl glutamic acid and 50 perg g 81 8'32cent N-palmitoyl glutamic acid were added in the 10 0 1:24 0:04 amountof 20 ug/ml or 50 ug/ml at 0, 5, 8 or 12 hours 7 2 098 l 6 cultivationtime. After 12 hours cultivation in the same 3 2 manner as in Example 1,the amounts of glutamic acid 3 -3Z 8%? as shown 1n Table 4 were found Inthe culture broths. 3 0 0560 0.38 Infected 5 0 0.870 0.61 Table 4 10 01.17 0.30 i i 8833 18% 0 5 8 12 Hours Hours Hours Hours I0 2 H5 (g/dl)(g/ (g/ l) tgl n 20 #g/ml 1.69 1.68 1.00 0.80 50 1.55 1.60 1.08 0.81EXAMPLE 6 An aqueous culture medium was prepared to con- 35 tain, perdeciliter, 10 g glucose, 0.1 g KH PO 0.04 g EXAMPLE 5 MgSO '7H O, 0.2 mgferrous ion, 0.2 mg manganese Brevibacterium lactofermentum ATCC 13869was 100 mg (as mtrogen) P P acld'hydfolyzatei cultured in the presenceof phage L (phages number g (Nlihhsoh 5 thlamme'HCl: and adlusted atcells number 73 x -2 or in the presence of pH 8.0. 20 Ml batches wereplaced 1n 500 ml flasks, phage S1 (phages number cells number 42 X 102)and to each flask was added the amount of N-stearoyl Phage S is reportedin Agricultural and Biological glutamic aCIdFhOWn m Table ChemistryJapan vol 31 N0 7 8614367 (1967) The Brevibacterium lactofermentumFERM-P 1711 was medium ernployed was i Same as in Example 1 inoculatedin each flask and cultured at 30C for 72 cept for the amount of biotin(shown in Table 5). N- 45 l Shakmg palmitoyl glutamic acid was added tothe medium in Mlcroblal growth and Lflysme accumulated were the amountof O 2 or #g/mL termined and are shown in Table 7.

Growth of Brevibacterium lactofermentum ATCC Table 7 13869 wasdetermined by measuring optical density of 26 times dilution at 562 mu.Glutamic acid in the cul- 50 PhagesNoJCells N-stearoyl Growth Lysineture broth was also determined. The results with phage :322:12 iaccumula'ed L, and with phage S are shown in Tables 5 and 6, re- (Lg/m1)(X 1/26) (g/dn spectlvely.

0 0 1.10 3.80 Table 5 0.2 1.05 3.92 2 1.08 3.59 20 1.09 3.62 PhageBiotin N-palmitoyl Growth Glutamic acid 200 1.05 3.73 glutamic acidaccumulated X -3 0 02 013 ag/1) wig/ml) l 0.2 0.32 0.56 2 0.58 1.20 0 00.406 1.11 20 1.10 3.63 I O 0.770 1.63 200 03 3 7 Not 2 0 0.950 1.23infected 3 0 0.975 0.64 5 0 1.04 0.06 10 0 1.24 0.04

EXAMPLE 7 2 50 0.90 1.62 3 50 0.95 0.81 An aqueous culture medium wasprepared to con- 28 1% 862 tain, per deciliter, 8 g glucose, 0.02 g KHPO 1.5 g. 3 O 0:300 NH NO 0.2 g CaCl 0.2 mg ferrous ion, 0.2 mg man- 50.16 ganese ion, 4 ml/dl soy-protein acid hydrolyzate, and' 0.l g RNA ofyeast. 20 Ml batches of the medium were placed in 500 ml flasks, and toeach flask was added the amount of N-oleoyl glutamic acid potassium saltshown in Table 8. The flasks were sterilized with steam.

Each flask was inoculated with Bacillus subtilis FERM-P 2107, and washeld at 34C for 72 hours with shaking. A phage of FERM-P 2107 was addedafter 12 hours cultivation. Growth of FERM-P 2l07 and guanosineaccumulated in the culture broth were determined and are shown in Table8.

An aqueous culture medium was prepared to contain, per deciliter, l gbouillon, l g peptone, 0.5 g soyprotein and 5 g soluble starch, andadjusted at pH 7.0. 50 Ml batches were placed in 500 ml flasks. To eachflask was added the amount of N-oleoyl homocysteic acid shown in Table9. The flasks were sterilized with steam, and inoculated with Bacillussubtilis FERM-P 305. Cultivation was carried out at 3 l .5C for 48 hourswith shaking. After 6 hours cultivation l P.F.U. of phages/ml were addedto the medium.

The protease activities shown in Table 9 were found in the culturebroth.

6 Table 9 Phage N-olcoyl lrotcase homocysteic acid activity sodium salt(#g/ml) (units/dl) 0 600 0.1 1,340 Added 0 2,500 O.l 2,640 Not addedWhat is claimed is:

1. A method for limiting damage resulting from the presence of abacteriophage in a fermentation medium, which comprises effecting thefermentation in a medium containing an amount of at least oneN-acylamino derivative of glutamic acid, glutamine, homocysteic acid,metallic salts thereof or mixtures thereof which is effective to inhibitthe production of bacteriophage in the fermentation medium, said acylradical of said N- acylamino group being characterized by containing analkyl or alkenyl moiety having from about 16 to 18 carbon atoms.

2. A method as in claim 1, wherein the amount of N- acylamino derivativeis less than 50 ug/ml.

3. A method as in claim 1, wherein the fermentation medium contains aglutamic acid producing strain of the genus Brevibacterium.

4. A method as in claim 1, wherein the fermentation medium contains alysine producing strain of the genus Brevibacterium.

5. A method as in claim 1, wherein the fermentation medium contains aguanosine producing strain of the genus Bacillus.

6. A method as in claim 1, wherein the fermentation medium contains aprotease producing strain of the genus Bacillus.

7. A method as in claim 1, wherein the N-acylamino acid isN-stearoylglutamic acid.

8. A method as in claim 1, wherein the N-acylamino acid is N-oleoylglutamic acid.

1. A METHOD FOR LIMITING DAMAGE RESULTING FROM THE PRESENCE OF ABACTERIOPHAGE IN A FERMENTATION MEDIUM, WHICH COMPRISES EFFECTING THEFERMENTATION IN A MEDIUM CONTAINING AN AMOUNT OF AT LEAST ONEN-ACYLAMINO DERIVATIVE OF GLUTAMIC ACID, GLUTAMINE, HOMOCYSTEIC ACID,METALLIC SALTS THEREOF OR MIXTURES THEREOF WHICH IS EFFECTIVE TO INHIBITTHE PRODUCTION OF BACTERIOPHAGE IN THE FERMENTATION MEDIUM, SAID ACYLRADICAL OF SAID N-ACYLAMINO GROUP BEING CHARACTERIZED BY CONTAINING ANALKYL OR ALKENYL MOIETY HAVING FROM ABOUT 16 TO 18 CARBON ATOMS.
 2. Amethod as in claim 1, wherein the amount of N-acylamino derivative isless than 50 Mu g/ml.
 3. A method as in claim 1, wherein thefermentation medium contains a glutamic acid producing strain of thegenus Brevibacterium.
 4. A method as in claim 1, wherein thefermentation medium contains a lysine producing strain of the genusBrevibacterium.
 5. A method as in claim 1, wherein the fermentationmedium contains a guanosine producing strain of the genus Bacillus.
 6. Amethod as in claim 1, wherein the fermentation medium contains aprotease producing strain of the genus Bacillus.
 7. A method as in claim1, wherein the N-acylamino acid is N-stearoylglutamic acid.
 8. A methodas in claim 1, wherein the N-acylamino acid is N-oleoyl glutamic acid.